This proposal is focused on the transcriptional regulatory mechanisms that control BMP-2 expression in chondrocyte populations and the effects of these regulatory mechanisms on the chondrocytic phenotype. The proposed research addresses the overall hypothesis that epigenetic mechanisms act on distant enhancer sequences to regulate chondrocyte-specific BMP-2 expression. The proposed experiments are designed to identify the distant enhancer sequence(s) that control BMP-2 expression in chondrocytes, define the lineage-specificity of this enhancer activity and assess the role of this distant enhancer sequence in phenotypic and biosynthetic alterations that occur during cartilage aging, arthritic disease and MSC chondrogenic differentiation. Experiments in Aim 1 will use a ChIP-based strategy targeting the histone acetyltransferase, p300, and mono-methylated lysine 4 of histone 3 to identify the transcriptionally active BMP-2 enhancer sequences in articular chondrocyte populations. Both these epigenetic markers are closely linked with active enhancer sequences. These experiments address the hypothesis that BMP-2 expression in articular chondrocytes is regulated by a 5'distant enhancer. Enhancer activity will be assessed using standard reporter assays. Aim 2 experiments will assess the lineage-specificity of the chondrocytic BMP-2 enhancer and will test the hypotheses that 1. The distant BMP-2 enhancer identified in articular chondrocytes is active in all chondrocytic populations, and 2. The chondrocytic BMP-2 enhancer is phenotype-specific and will not stimulate BMP-2 expression in other articular/per-articular tissues. These experiments will determine whether the enhancer identified in articular chondrocytes is also active in other chondrocyte populations and is specific for the chondrocytic lineage. Aim 3 experiments will assess the epigenetic status of the BMP-2 distant enhancer in the contexts of articular cartilage 'aging', arthritis and during stem cell chondrogenesis. These experiments will test the hypothesis that alterations in chondrocyte BMP-2 expression occur through epigenetic changes in enhancer status in these three clinically important contexts. Collectively, the experiments will determine the role of epigenetics in regulating BMP-2 expression in chondrocytes, during aging, in osteoarthritis and during chondrogenic differentiation. PUBLIC HEALTH RELEVANCE: Cartilage is a highly specialized connective tissue that serves as a temporary scaffold for bone formation during skeletal development and fracture repair, and also persist throughout our lifetimes on the surfaces of joints, as articular cartilage. Several lines of evidence indicate that robust BMP-2 expression is mandatory for the normal homeostatic and developmental activities of chondrocyte populations. Recent genomic studies have confirmed that cell/tissue- specific gene expression is regulated by highly conserved enhancer sequences, often very distant from the gene coding region itself. BMP-2 appears to be regulated in this manner. The proposed studies will determine the mechanism by which chondrocytes regulate BMP-2 expression in a cell/tissue-specific manner and investigate the role of this regulatory mechanism in the reduced BMP-2 expression seen during aging, in arthritis and during stem cell chondrogenic differentiation.